PROJECT SUMMARY Hematopoietic stem cells (HSCs) give rise to all terminally differentiated cells in the blood. The ability of HSCs to reconstitute these blood cell lineages for life underlies the efficacy of bone marrow transplantation therapy for treatment of various blood disorders, including leukemias, anemia, and autoimmunity. Although this is an established and effective treatment, two-thirds of patients in need of a transplant lack a matched donor. Therefore, alternative sources of therapeutic HSCs would be a boon to the field. Human pluripotent stem cells (hPSCs) represent a potential source for cell-based therapies, including the derivation of patient-specific transplantable HSCs, which would additionally circumvent immune rejection and alloreactivity, both major issues in the clinic. The proposed collaborative research leverages the expertise of two Principal Investigators with complementary research interests and skills in stem cell biology, zebrafish genetics and development, hematopoietic development, and Wnt biology and biochemistry. Using zebrafish as a model organism, they seek to identify and characterize the molecular cues, in particular Wnt signaling, that direct hematopoietic development during early embryonic stages. As indicated by their ongoing collaborative studies, the Wnt signaling pathway plays a critical role in the development of HSCs. The proposed studies will build on the observation that Wnt9a is specifically required for HSC formation, emergence and expansion by (1) determining the cognate Wnt9a receptor(s) by genetic, cell biological and biochemical means, (2) generating knockout and conditional knockout alleles of Wnt9a and candidate receptors and thereby assessing their spatial and temporal requirements, (3) identifying the Wnt producing and Wnt-responding cells, and (4) dissecting the gene expression and epigenetic changes that accompany HSC development. The long-term goal of these studies is to gain a better understanding of how HSCs develop in the embryo. Given that fundamental developmental processes, including blood formation, are highly conserved from fish to humans, the discoveries made in the course of the proposed work will directly inform current methods and protocols to derive HSCs from hPSCs. Therefore, successful completion of this research will have a profound impact on HSC derivation and expansion, and thereby will be instrumental in overcoming current obstacles to the effective treatment of diseases requiring bone marrow transplant therapy.